MICA: Finding new treatments for failed resolution of inflammation using zebrafish models.

Neutrophils are a type of white blood cell that are important in fighting infection and healing wounds (in a process called inflammation). We know that neutrophils need to be removed when their job is done or they can cause damage. The mechanisms of neutrophil removal are particularly important in a delicate organ such as the lung where failure can mean permanent lung damage as is seen in Chronic Obstructive Pulmonary Disease (COPD), a common disabling disease with no cure. We do not understand how neutrophils know when the job is done and how this translates into signals to remove neutrophils from their place of work - the inflammatory site. In my lab, I have developed tools in zebrafish that allow me to see neutrophils as they fight infection, heal wounds and importantly as they are removed during healing (or "resolution") of inflammation. These zebrafish can be genetically modified to express glowing (fluorescent) proteins that label neutrophils or to change the genes controlling inflammation. This allows us to get a much better understanding of the processes and controls of inflammation resolution. With these models, we have seen how neutrophils can be removed in 2 ways, by moving away from the site of inflammation or by dying there and being removed by another type of immune cell (macrophages).
In this programme, I propose to use zebrafish models and human neutrophils to understand the signals keeping neutrophils at sites of inflammation, which I call "retention signals". I have identified a drug that works by causing neutrophils to leave sites of inflammation and I will investigate how this changes the retention signals that neutrophils use to keep them at inflammatory sites. I will then study the downstream signals within neutrophils that act to turn these signals into changes in behaviour of the neutrophils. From recent studies in my lab, I have identified two important pathways that might be key regulators of retention signalling. The first is an enzyme, Serum and Glucocorticoid regulated kinase-1 (SGK1) that seems to act to retain living neutrophils at sites of inflammation. I will explore the potential of this kinase and related molecules to control inflammation resolution. Since this is a good drug target, this aim will be explored in collaboration with GlaxoSmithKline who are working with me to identify drug candidates targeting this molecule. Finally, a gene called Kalirin was identified in a genetic screen in my lab as a regulator of inflammation resolution. Kalirin is a protein normally controlling the direction of nerve cell growth, but it is present in neutrophils where it might control neutrophil movement. I will explore the possibility that Kalirin is involved in retention signalling and see whether it might be a suitable target for drug discovery.
Taken together these approaches will uncover new aspects of the control of inflammation resolution and identify a number of targets for drug discovery. I hope this will ultimately find new ways to treat inflammatory diseases.

Diseases of failed inflammation resolution such as COPD are major causes of morbidity and mortality. Currently no therapies target the key effector cell, the neutrophil, to drive inflammation resolution. Targeting neutrophil recruitment leaves tissues unprotected against infection, and new approaches are needed to remove unwanted neutrophils without impacting host defence. We are in the midst of a revolution in neutrophil biology, in which neutrophils are recognised to be more long-lived, with active roles in programming adaptive immunity and regulating immune homeostasis. Neutrophils are now recognised to have a number of potential fates in addition to apoptosis. Neutrophils can reverse transmigrate back into the circulation in humans and mice, they can be lost into exudates in the mammalian lung or they can reverse migrate within tissues to dissipate the inflammatory burden - shown in zebrafish and with human neutrophils.
I hypothesise that neutrophils are retained at inflammatory sites by "retention signals" that delay reverse migration, and that neutrophil sensitivity to these signals is mediated through a pathway requiring Serum and Glucocorticoid-regulated Kinase-1 (SGK1) and Kalirin, providing a series of new targets for drug discovery. In this Programme, I will identify the nature of retention signals and how these overlap with survival signals, then explore the molecular mechanism of action of Tanshinone IIA (identified by drug screening in my lab) which drives reverse migration. I will define the functional neutrophil kinome and exploit novel kinase inhibitors and kinase targets for the selective removal of inflammatory neutrophils, using SGK1 as a prototypic downstream signal important in retention signalling. Finally, I will define the role of Kalirin (identified by genetic screening in my lab), as a regulator of neutrophil retention signalling.

The mechanisms regulating inflammation resolution are of fundamental importance in immunology. Very few targets with critical roles in inflammation resolution have been identified to date. The discoveries from this project will therefore significantly enhance the knowledge economy with new scientific advancement, as described in 'academic beneficiaries".
There is considerable interest from the pharmaceutical industry in discovering regulators of inflammation resolution, which are fundamental in a range of disease settings. In my own clinical speciality of respiratory medicine, Chronic Obstructive Pulmonary Disease is a major cause of morbidity and mortality which lacks effective treatments. Many examples exist in all clinical specialities. We anticipate that the unique nature of the mechanisms investigated in this Programme will make them attractive targets for anti-inflammatory therapeutics. Protection of IP for these targets as they are discovered will bring significant economic gains to UK plc. Integration with the Pharma industry will allow rapid drug development, building on existing collaborations such as the MICA collaboration with GlaxoSmithKline for Aim 2 of this proposal.
It is my ultimate aim that drugs identified in my research programme find their way into my clinical practice to treat patients who I currently cannot offer effective treatments. This programme will deliver successful identification of drug targets and identification of lead candidates which I hope will ultimately have an impact on patient care. The approaches I have developed are suitable for drug repurposing, and identification of compounds such as Tanshinone IIA (used in China to treat cardiovascular disease) shows how this might be possible. Experimental Medicine approaches are in development to take forward such advances towards clinical use. The project will validate approaches for the identification of new small molecular inhibitors of retention signalling in inflammation. This leads to obvious longer-term commercial opportunities to develop the technology to a higher throughput level, and to engage industrial partners in developing new therapies. Via existing and new links, we will encourage Pharma investment in this programme, and develop IP sharing arrangements to ensure mutual benefit from emerging knowledge and know how.
The advances in knowledge, and potential for driving drug development will ultimately impact on quality of life, health and well-being. Avoidance of dysregulated inflammation is a prerequisite for healthy ageing.
The project uses cross-disciplinary approaches from mammalian cell biology, zebrafish models and molecular biology. These methodologies will be used to develop and make use of innovative approaches to the study of inflammation in vivo. The project will contribute to new expertise in developing these unique tools to address biological questions by a systematic, and ultimately, high throughput approach. The project will strengthen links between different disciplines and forge a greater understanding of how we can engage, complement and enhance research for the future.
This proposal will deliver highly trained researchers offering unique skills. The PDRAs will combine skills in fish models and state of the art in vivo microscopy, in parallel with human primary cell-based assays. They will develop distinctive skills in generating new datasets and new approaches to understanding biological problems. This expertise will provide transferable skills to other non-academic beneficiaries, but will also be used to train researchers from other groups in our methodologies.
The PDRAs and myself are actively involved in public engagement and broader dissemination, with regular school visits and high-level involvement with public exhibitions such as Royal Society Summer Science exhibition, and the University of Sheffield Festival of the Mind.